Platelets are anucleated cells that are the primary cells responsible for stopping bleeding. Blood platelets are approximately 3 microns in size and circulate in the blood stream as disc shaped cells that upon activation by either tissue injury or exposure to a foreign material undergo physiological changes that lead to aggregate formation at the site of injury or foreign material. Blood platelets circulate at approximately 250,000 to 350,000 platelets per microliter of whole blood. Upon activation, platelets change shape from a disc to a sphere and form pseudopodia elongations.
The normal platelet response to initiate cessation of bleeding is to undergo a shape change, attach to the surface, and release intraplatelet components that act to provide an autocatalytic recruitment of more platelets. With the recruitment of additional platelets, a platelet plug or aggregate mass forms. The aggregate mass evolves from a single platelet of only 3 microns in size to a mass on the order of millimeters in size. The platelet mass additionally recruits and participates with the plasma coagulation proteins. The plasma coagulation proteins undergo a cascade of events involving 13 enzymes and cofactors, which leads to the activation of plasma fibrin to form a fibrin clot.
It is useful here to briefly summarize the biochemical events of hemostasis (the cessation of bleeding). Normal intact endothelium does not initiate or support platelet adhesion (although in certain vascular diseases platelets may adhere to intact endothelium). Vascular injury, however, exposes the endothelial surface and underlying collagen. Following vascular injury, platelets attach to adhesive proteins such as collagen via specific glycoproteins on the platelet surface. This adhesion is followed or accompanied by platelet activation, where platelets undergo a shape change from a disc shape to a spherical shape with extended pseudopodia. At this time, the platelet release reaction also occurs. The platelets release biologically active compounds stored in the cytoplasmic bodies that stimulate platelet activation or are otherwise involved in clotting reactions. These include ADP, serotonin, thromboxane A2, and von Willebrand factor. Thromboxane A2 is a potent inducer of platelet secretion and aggregation. It is formed by the enzyme cyclooxgenase, which is inhibited by aspirin, among other drugs.
Following activation, glycoprotein IIb and IIIa (GPIIbIIIa) receptors on the surface of the platelets undergo a conformational change from a relatively inactive conformation to an activated form. GPIIbIIIa receptors mediate the adhesion of more platelets by adhering to the circulating plasma protein fibrinogen, which serves as a bridging ligand between platelets. The adhesion and aggregation of platelets constitutes primary hemostasis.
Secondary hemostasis stabilizes the platelet mass by forming a fibrin clot. The fibrin clot is the end product of a series of reactions involving plasma proteins. The process is known as blood coagulation. Among the plasma proteins involved are the activated forms of the clotting factors II, VII, IX, X, XI, and XII (the activated forms have an “a” following the Roman numeral, e.g., factor IIa). The activated forms of these proteins are serine proteases.
Fibrin is formed from fibrinogen, a large circulating plasma protein, by specific proteolysis. In the process, the protein thrombin (factor IIa) is consumed. Fibrin monomers next spontaneously associate to form polymers and form a loose reinforcement of the platelet plug. Fibrin polymers are then cross-linked by certain enzymes. The fibrin polymer also traps red cells and white cells to form a finished clot.
Under normal conditions of hemostasis, the individual experiencing bleeding benefits from the ability of platelets to change shape, adhere, spread, release chemical messengers and activators, aggregate, and assemble with fibrin. This series of events stops bleeding at the site of injury and initiates the process of wound healing.
But platelet activation and clot formation can also place a person at risk of pathological cardiovascular events. For example, venous blood clot formation in the legs, a condition known as deep vein thrombosis, creates the risk that the blood clots could embolize (break apart) and result in clot entrapment in the lungs or the brain, causing pulmonary embolisms and stroke-related conditions. Platelet activation and fibrin formation in other locations in some persons create aggregates and small clots in the arterial circulation that can also lead to embolization and strokes.
In addition to age and genetic and lifestyle risk factors, implanted medical devices in the blood stream also place patients at greater risk of clot formation and embolization. Each year, approximately 500,000 heart valves are implanted in the United States. Although biomaterial advancement has somewhat reduced the risk of thrombosis (clot formation), all patients with mechanical heart valves are at increased risk of clot formation, embolization, and stroke.
Arterial stents are another type of device placed in the circulatory system that place patients at risk from platelet activation. Arterial stents are placed in clogged coronary and carotid arteries to provide oxygen to cardiac tissue. They are typically around 5 mm in diameter and are made from stainless steel or other materials. Due to the introduction of a foreign material in the blood stream, platelets can become activated and attach to the wall of the stented vessel. This leads to reocclusion (restenosis) of the stented vessel, which is a very significant risk in patients with arterial stents. Restenosis in the first 28 days is reported in 0.5 to 8% of persons receiving stents.
In an effort to reduce the risk of embolization and restenosis, patients receiving heart valves or arterial stents are commonly placed on anti-coagulant or platelet-inhibiting medications before, during, and after the procedures.
Current platelet inhibiting drugs fall into three groups: (1) aspirin-related drugs, which inhibit the platelet cyclooxygenase enzyme, thus reducing production of thromboxane A2, which is a platelet activator; (2) ADP-receptor inhibiting drugs, which block a surface membrane receptor on the platelets that is involved in the activation process; (3) monoclonal antibodies that block GPIIbIIIa receptors on the platelet surface. The GPIIbIIIa receptor binds the plasma coagulation factor fibrinogen, which is involved in both aggregation and in forming a fibrin clot.
All three approaches are effective in reducing platelet activation, however no intervention is successful on all patients. Aspirin is the least expensive. But the appropriate dose varies unpredictably from person to person, and up to 30% of individuals on long-term aspirin therapy do not achieve inhibition of platelet adhesion. The ADP-inhibiting drugs are more expensive than aspirin, but are gaining popularity. However, as with aspirin, the required dose and duration of therapy varies, and a large variation in platelet adhesion characteristics in patients on the drugs exists. The GPIIbIIIa-inhibiting drugs are argued to provide the greatest platelet inhibition, but they are very expensive and still suffer from patient-to-patient variability in dosing and effectiveness. Other medications are likely to emerge, but all will probably still have the patient-to-patient variability seen with other approaches.
The failure to determine the proper dose and medication to inhibit platelets can have a great cost in money, and can cause unnecessary morbidity and death. For example, patients on anti-GPIIbIIIa drugs have been reported to have from a 5.8 to 11.2% incidence of adverse reactions in the first 28 days after stenting. The adverse reactions were defined as death, myocardial infarction, or urgent need for reintervention with angioplasty procedures. The risk was even higher when patients were not treated with the drugs. (New England J. Med. 330:956-961, 1994; New. England J. Med. 336:1689-96A, 1997; Lancet 349:1429-35, 1997.)
Thus, anti-platelet drugs have a large patient-to-patient variability and many patients are refractory to some anti-platelet drugs. A method is needed to monitor platelet function so the proper dose of an anti-platelet drug for a particular patient can be determined, and so a physician can determine whether a particular patient is refractory to one anti-platelet drug but responsive to another.
No reliable point-of-care method currently exists to specifically determine if platelet adhesion and aggregation have been inhibited. Thus, there is a need for a method and a device to measure platelet function, and preferably to measure platelet adhesion and aggregation as part of the measurement of platelet function. The need to measure platelet function is particularly acute in patients receiving arterial stents or other cardiovascular devices, and in other persons at risk of adverse cardiovascular events. Such a method would allow an attending physician to ensure that platelet function has in fact been inhibited in a patient at risk, and to adjust pharmacologic parameters prior to implanting a cardiovascular device, which will reduce the risk of adverse events associated with platelet initiation of clot formation.
Another need to monitor platelet function arises in platelet transfusions. Platelets are harvested and used in platelet transfusions to support patients at risk of bleeding. However, platelet storage poses problems not found with the storage of whole blood or other components. Whole blood, red and white cells may be stored at 4° C. for weeks. However, platelets will aggregate in cold storage and when allowed to settle. Therefore, the standard means of storing platelets is at room temperature with gentle agitation. Even under these conditions, platelets lose function by about 5 days. Thus, methods and devices for monitoring platelet function are also needed to determine whether stored platelets have adequate activity to be transfused into patients.
Another need to monitor platelet function exists to test patients undergoing a medical or dental procedure for their risk of excessive bleeding during the procedure.
Accordingly, a need exists for a method to measure platelet function. Preferably, the method would monitor platelet adhesion and aggregation. Preferably, the method would monitor platelet function specifically, separately from the other aspects of clotting such as blood coagulation. Preferably, the method would be inexpensive. Preferably, the method would not depend upon platelet activation by any particular chemical platelet activator or group of chemical platelet activators. Preferably, the method could be used on whole, unprocessed blood, and could produce results quickly (e.g., be used at the bedside, during a physician visit, or during a medical procedure to provide a result almost immediately). Devices to monitor platelet function are also needed.